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Hepatic cyclooxygenase2 expression protects against diet induced steatosis, obesity and insulin resistance. ●1Daniel E. Francés, ●2Omar Motiño, ●2Noelia Agrá, 2,3Águeda GonzálezRodríguez,

1Ana FernándezÁlvarez, 5Carme Cucarella, 4,6Rafael Mayoral, 2Luis CastroSánchez,

2Ester GarcíaCasarrubios, 2,4Lisardo Boscá, 1Cristina E. Carnovale, 4,5Marta Casado,

♦2,3Ángela M. Valverde and ♦2,4Paloma MartínSanz.

Keywords: COX2, hepatocyte, HFD, obesity, IR.

Running title: COX2 in insulin resistance

Affiliations:

1Institute of Experimental Physiology (IFISECONICET), Suipacha 570, 2000 Rosario,

Argentina. 2Institute of Biomedical Research Alberto Sols, CSICUAM, Arturo Duperier

4, 28029 Madrid, Spain; 3Networked Biomedical Research Center on Diabetes and

Metabolic Diseases (CIBERdem), Monforte de Lemos 35, 28029 Madrid, Spain;

4Networked Biomedical Research Center on Hepatic and Digestive Diseases

(CIBERehd), Monforte de Lemos 35, 28029 Madrid, Spain; 5Biomedical Institute of

Valencia, IBVCSIC, Jaume Roig 11, 46010 Valencia, Spain; 6Department of Medicine,

University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

♦ Corresponding authors.

These two authors share senior authorship (♦)

Dr. Paloma MartínSanz or Dr. Ángela M. Valverde, Institute of Biomedical Research

Alberto Sols, CSICUAM. Madrid. Arturo Duperier, 4 28029 Madrid, Spain Tel

34914972746, 34915854497 Fax: 34915854401; email: [email protected] or [email protected]

● These authors contributed equally to this work

Diabetes Publish Ahead of Print, published online November 24, 2014 Page 3 of 47 Diabetes

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Figures: 5

Tables: 3

Word count abstract: 198

Word count manuscript: 3865

Abbreviations

COX2, cyclooxygenase2; PGE2, prostaglandin E2; RCD, regular chow diet; HFD, high fat

diet; Ins, Insulin; DFU,(5,5dimethyl3(3fluorophenyl)4(4methylsulfonyl) phenyl2(5H)

furanone); PTP1B, protein tyrosine 1B; Cpt1a, carnitine palmitoyltransferase 1

liver isoform; Scd1, stearoylCoA desaturase 1, TG, ; Pgc1α, PPARγcoactivator

1α; Acox1, palmitoylCoA oxidase; Pparα, peroxisome proliferatoractivated receptor alpha;

Pparγ, peroxisome proliferatoractivated receptor gamma; Acaca, acetylCoA carboxylase 1;

Fasn, fatty acid synthase; Lipc, hepatic triacylglycerol ; Lipe, hormonesensitive lipase;

Pnpla2, patatinlike domaincontaining protein 2; Cd36, fatty acid ;

mpges1, microsomal prostaglandin E synthase1; Bcl2, Bcell lymphoma 2; UCP1,

uncoupling protein1; Dio2, type II iodothyronine deiodinase; Prdm16, PR domain containing

16; ALT, alanine transaminase; AST, aspartate transaminase, NEFAs, nonesterified free fatty

acids; HOMA, homeostasis model assessment of insulin resistance; IR, insulin resistance; IL6,

interleukin 6; IL1β, interleukin 1β; TNFα; tumor necrosis factor α; EE, energy expenditure;

RER, respiratory exchange ratio; SVF, stromal vascular fraction.

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ABSTRACT

Accumulation evidence links obesityinduced inflammation as an important contributor to the development of insulin resistance which plays a key role in the pathophysiology of obesityrelated diseases such as type 2 diabetes mellitus and non alcoholic fatty liver disease. Cyclooxygenase (COX)1 and 2 catalyze the first step in prostanoid biosynthesis. Since adult hepatocytes fail to induce COX2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX2

(hCOX2) in hepatocytes protects against adiposity, inflammation, and hence insulin resistance induced by high fat diet as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin/leptin ratio and decreased levels of proinflammatory cytokines together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX2 transgenic mice exhibited increased whole body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptormediated Akt phosphorylation in hCOX2Tg. In conclusion, our results point to COX2 as a potential therapeutic target against obesityassociated metabolic dysfunction.

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INTRODUCTION

Insulin resistance (IR) plays a key role in the pathophysiology of obesityrelated

diseases such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). IR is

the key primary defect underlying the development of type 2 diabetes and is a central

component defining the metabolic syndrome. It has been demonstrated that IR is

associated with a state of chronic lowgrade inflammation, and it is assumed that

inflammation contributes in a major way to its development (1). Besides the fact that IR

is characterized by complex interactions between genetic determinants and nutritional

factors, it is recognized that mediators synthesized from immune cells and adipocytes

are involved in the regulation of insulin action (2). Insulin acts in target cells by binding

to its specific receptor and activating a cascade of intracellular signaling events that are

altered in inflammationassociated IR (3).

Inflammation induces the expression of a variety of proteins including

prostaglandinendoperoxide synthase2, also known as cyclooxygenase (COX)2, which

catalyzes the first step in prostanoid biosynthesis. COX2 is induced by a variety of

stimuli such as growth factors, proinflammatory mediators, hormones and other

cellular stresses (4). However, adult hepatocytes fail to induce COX2 regardless of the

proinflammatory stimuli used and only under prolonged aggression as a result of the

drop of C/EBPα (5) or after partial hepatectomy (PH), COX2 is induced (6). On the

other hand, Kupffer, stellate, hepatoma mouse liver cells and fetal hepatocytes retain the

ability to express COX2 upon stimulation with LPS and proinflammatory cytokines

(7).

Previous work has implicated COX2 in the pathogenesis of obesity and IR.

Hsieh et al. (810) reported an improvement of muscle and fat insulin resistance in rats

fed with fructose or high fat diet (HFD) treated with COX2 inhibitors. However, Coll Diabetes Page 6 of 47

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et al. found that COX2 inhibition exacerbates palmitateinduced inflammation and IR in skeletal muscle (11). Our previous results (12) performed in a model of transgenic mice constitutively expressing human COX2 in hepatocytes (hCOX2Tg) demonstrated a protective role of COX2 against liver apoptosis induced by streptozotocin (STZ)mediated hyperglycemia. These findings prompted us to screen the role of COX2 expression in hepatocytes in a model of IR and altered energy homeostasis induced by HFD. The present study has demonstrated that expression of

COX2 in hepatocytes protects against steatosis, adiposity, inflammation and hepatic insulin resistance, in hCOX2Tg mice under HFD by improving insulin sensitivity and glucose tolerance, enhancing Akt and AMPK phosphorylation, decreasing protein tyrosine phosphatase1B (PTP1B) protein levels and increasing thermogenesis and energy expenditure.

RESEARCH DESIGN AND METHODS

Animal experimentation. hCOX2Tg mice (2530g body weight; 3 months) on a B6D2/OlaHsd background were used in this study along with corresponding agematched wildtype (Wt) mice (13).

Animals were treated according to the Institutional Care Instructions (Bioethical

Commission from Spanish Research Council, CSIC, Spain). hCOX2Tg mice and their corresponding Wt littermates were generated by systematic mating of Tg mice with

B6D2F1/OlaHsd Wt mice in our animal facilities for more than seven generations.

Insulin signaling studies. 6 hfasted Wt and hCOX2Tg mice were i.p. injected with

0.75 U/kg human recombinant insulin, and sacrificed 15 min later. Liver was removed, and RNA and total protein extracts were prepared as previously described (14).

Induction of liver steatosis and obesity by high fat diet (HFD). Wt and hCOX2Tg mice were fed with regular chow diet (RCD, A0410, Panlab, Barcelona, Spain) or a Page 7 of 47 Diabetes

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42% highfat diet (HFD) (TD. 88137, Harlan Laboratories, Madison, WI, USA) ad

libitum for 12 weeks. Some (n=7) of the hCOX2Tg mice fed a HFD were treated i.p.

with 10 mg/kg /day DFU, a COX2 selective inhibitor, five times in a week during all

the treatment. During HFD treatment, body weight, food and water intake were

recorded every two days. After 12 weeks of treatment, animals were sacrificed and

liver, epididymal WAT (eWAT), inguinal WAT (iWAT) and interscapular brown

adipose tissue (BAT) were snapfrozen in liquid nitrogen and stored at 80°C or

collected in a solution containing 30% sucrose in PBS or fixed in 10% buffered

formalin. Plasma was obtained from the inferior vena cava.

Metabolic measurements. Metabolic measurements were performed at the beginning of

HFD treatment and at the end of week 12. Mice were subjected to insulin tolerance test

(ITT) and glucose tolerance test (GTT). For ITT, after 6 h fasting, mice were i.p.

injected 0.75 U/kg of human recombinant insulin. Blood glucose was measured from

the tail vein at 0, 15, 30, 60 and 90 min. For GTT, 16 h fasted mice were i.p. injected 2

g/kg glucose and blood samples were taken at 0, 30, 60 and 90 min. Glucose levels were

measured with an AccuCheck Glucometer (Roche). The HOMAIR (homeostasis

model of assessment of insulin resistance), an index of whole body insulin resistance at

basal state, was calculated by HOMAIR=(FPIxFPG)/22.5; where FPI is fasting plasma

insulin concentration (mU/l) and FPG is fasting plasma glucose (mmol/l).

Indirect calorimetry. At the end of week 12, independent groups of Wt and hCOX2Tg

mice (n=4) were analyzed by indirect calorimetry in a PhenoMaster System (TSE

systems, Bad Homburg, Germany). Mice were acclimated to the test chamber for at

least 24 h and then were monitored for an additional 48 h. Food and water were

provided ad libitum in the appropriate devices and measured by the buildin automated

instruments. Volumes of consumed O2 (VO2) and eliminated CO2 (VCO2) were taken Diabetes Page 8 of 47

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every 10 min. Respiratory exchange ratio was calculated as: RER=VCO2/VO2. Energy expenditure was calculated as: EE=(3.815 + (1.23 x RER)) x VO2 x 1.44). Data are the average values obtained in these recordings.

Cold exposure experiments. Three month old Wt and hCOX2Tg mice (n=8) were acclimated at a temperature of 28ºC for one week. Then, mice were randomly divided into two groups, one group was exposed to 4ºC for 6 h in individual cages and the other group was maintained at 28ºC. At the end of the experiment, mice were sacrificed and

BAT, eWAT and iWAT depots were removed and frozen in liquid nitrogen for posterior assays.

Data analysis.

Statistical analyses were performed using the SPSS package v.17. Data are expressed as means ± S.E. Student’s ttest was applied whenever necessary, and statistical analysis of the differences between groups was performed by oneway ANOVA followed by

Tukey’s test. A p<0.05 was considered statistically significant.

RESULTS

COX2 transgenic mice are protected from HFDinduced hepatic steatosis, obesity and insulin resistance.

To explore if COX2 expression in hepatocytes affects glucose tolerance and insulin sensitivity in mice, we used our previously described transgenic mice (hCOX2Tg) that constitutively express human COX2 in hepatocytes under the control of the human

ApoE promoter and its specific hepatic control region (HCR), a unique regulatory domain that directs ApoE expression in liver (13), that also lacks macrophagespecific regulatory regions (ME.2 and ME.1) (15), ensuring exclusive expression in liver. PGE2, the main COX2derived prostanoid in liver, is three fold increased (Supplementary Fig.

1A and B) (13). The expression of COX2 in the liver of COX2Tg mice is comparable Page 9 of 47 Diabetes

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to the levels reached in fetal hepatocytes after stimulation with LPS (5; 7) and in liver

regeneration after PH (6; 16). Although, no differences in body weight gain were found

under RCD diet (Supplementary Fig. 1C), hCOX2Tg mice fed with a HFD diet did

not increase body weight as much as the corresponding Wt littermates and the eWAT

size and liver weight were significantly lower compared to those of the Wt (1.29±0.05*;

1.63±0.11 liver weight for hCOX2Tg and Wt respectively) (Fig. 1AC). Notably,

hCOX2Tg mice displayed a 20% lesser body weight gain without significant changes

in the initial body weight or in food intake (Fig. 1D). Interestingly enough, hCOX2Tg

mice treated with DFU exhibited an intermediate body weight value (Fig. 1C). To

assess the impact of HFD on glucose homeostasis, HFDfed mice were subjected to

GTT and ITT analysis (Fig. 1EF). Plasma glucose levels (mg/dl) were similar between

genotypes under HFD: 173.5±6.7; 159.2±12.1 and 177.3±21.2 for Wt, hCOX2Tg and

hCOX2Tg+DFU, respectively. However, under 42% HFD, hCOX2Tg mice

displayed enhanced insulin sensitivity (Figure 1E) and glucose tolerance (Figure 1F)

than the Wt. In addition, treatment of hCOX2Tg mice with DFU partially reversed the

beneficial effects on glucose homeostasis induced by COX2dependent prostaglandins.

ITT and GTT also revealed an enhanced insulin sensitivity and glucose tolerance in

hCOX2Tg vs. Wt mice under basal conditions (regular chow diet, RCD;

Supplementary Fig. 1D and E). The effect of COX2 expression in hepatic insulin

signaling was evaluated in primary hepatocytes. As shown in Supplementary Fig. 2A,

insulininduced insulin receptor and Akt phosphorylations were markedly increased in

hCOX2Tg hepatocytes. Furthermore, when Wt hepatocytes were treated with

prostaglandin E2 (PGE2) prior to insulin stimulation Akt phosphorylation was increased

and, conversely, when hCOX2Tg hepatocytes were pretreated with DFU, Akt Diabetes Page 10 of 47

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phosphorylation was decreased, indicating the specificity of COX2dependent prostaglandins in the modulation of insulin signaling (Supplementary Fig. 2B).

Chronic HFD treatment causes accumulation of lipids in the liver, a process leading to fatty liver disease. Under these nutritional conditions Wt mice developed severe steatosis (Fig. 2A), including massive accumulation of large lipid droplets and a significant increase in liver TG (Fig. 2B). hCOX2Tg mice were protected against liver steatosis, showing lesser and smaller lipid droplets whereas DFU administration partially reverted this effect exhibiting an intermediate liver steatosis but including lipids microvesicles (Supplementary Fig. 3A); steatosis score as defined by NASH

Clinical Research Network Scoring System Definitions (17) was lower in hCOX2Tg mice (Supplementary Fig. 3B).

COX2 transgenic mice are protected from hepatic inflammation and injury.

Since the increase in adipose tissue and inflammation has been linked to insulin resistance, we performed histological examination of eWAT and the results showed adipocyte hypertrophy with larger adipocytes in Wt mice vs. hCOX2Tg and, again,

COX2Tg mice treated with DFU resembled features of the Wt eWAT (Fig. 2C). A frequency histogram showed a shift to the left in adipocyte size in the hCOX2Tg mice with a significant increase in the number of cells and a decrease in the occupied area

(mean 1168±652, 624±356* for Wt and hCOX2Tg respectively); this process was partially reverted with the DFU treatment (748±863) (Fig. 2D and E). Moreover, as an estimation of body fat content, eWAT plus iWAT mass referred to body weight was lower in hCOX2Tg mice whereas tibia length did not change between Wt and hCOX

2Tg mice (Fig. 2F). These results indicate hypertrophy in eWAT from Wt mice.

Cytokine eWAT expression is shown in Fig. 2G. As expected, higher adiponectin levels Page 11 of 47 Diabetes

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were detected in hCOX2Tg mice together with a significant decrease in the pro

inflammatory markers TNFα and IL6.

Indeed, whereas plasma TGs were significantly elevated exclusively in obese Wt mice

after HFD, plasma cholesterol increased both in Wt and hCOX2Tg mice. Regarding

plasma free fatty acids (NEFA) levels, those in HFDfed Wt mice were higher than in

hCOX2Tg mice (Table I). Plasma insulin and leptin increased after HFD in Wt mice,

but lesser increases were found in hCOX2Tg mice. Furthermore, hCOX2Tg mice

were hypoleptinemic under RCD (Table I), and adiponectin levels were elevated (20%

increase) in hCOX2Tg mice vs. Wt controls under HFD. The adiponectin/leptin ratio

was higher in hCOX2Tg; however, HOMAIR was lower in hCOX2Tg mice under

HFD (Table I). hCOX2Tg mice treated with DFU partially prevented the beneficial

effects on plasmatic parameters induced by COX2dependent prostaglandins. In fact,

plasmatic PGE2 did not change with HFD however, as expected, PGE2 levels decreased

under DFU treatment (Table I). Hepatic injury was analyzed by measuring ALT and

AST activities and both increased by the effect of HFD to a lesser extend in

hCOX2Tg mice (Supplementary Fig. 4). HFD led to a chronic inflammatory profile,

which is believed to be critical for the development of glucose intolerance and insulin

resistance (18). Accordingly, hepatic mRNA levels of TNFα, Il1β and IL6, were

significantly enhanced in livers from HFDfed Wt mice, whereas hCOX2Tg mice

exhibited lower levels of these inflammatory markers (Table II).

After detecting the differences in NEFAs levels, High Resolution Magic Angle

Spinning Magnetic Resonance Spectroscopy (HRMAS MRS) was performed to

identify and quantify saturated fatty acids (Supplementary Fig. 5A and B). We found a

lower value of the pool of total saturated fatty acids in hCOX2Tg mice compared to

Wt, and this result was reversed in hCOX2Tg+DFU (Supplementary Fig. 5B). Diabetes Page 12 of 47

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To gain insight into the molecular mechanisms implicated in COX2 protection, we evaluated mRNA levels of transcription factors and genes involved in fatty acid oxidation, lipolysis and lipogenesis. In hCOX2Tg liver there was a significant increase of Ppara and Cpt1a under HFD indicating an enhanced fatty acid oxidation. In agreement, the expression levels of genes involved in fatty acid synthesis such as Pparg and Scd1 were markedly increased in Wt, but upregulation of these markers was lower in hCOX2Tg mice. Also, Cd36 expression was lower in hCOX2Tg. Moreover, gene expression of COX2 mediatedresponsive primary targets has been analyzed. As expected, hCOX2Tg mice showed an increase in hepatic mRNA levels of CyclinD1 and Bcl2, known target genes of COX2, and mPges1, the second key that couples with COX2 for the synthesis of PGE2 (Table III).

Increased energy expenditure in COX2 transgenic mice after HFD treatment.

To determine the cause of decreased adiposity and improved insulin sensitivity in hCOX2Tg mice, we examined food intake and energy expenditure (EE). The reduced adiposity of hCOX2Tg mice could not be explained by decreased food intake (Fig.

1D), but was associated with increased EE. To note, no differences were found in basal rectal temperature by a possible pyrogenic effect of PGE2 (Wt: 36.4±0.6; hCOX2Tg:

35.8±0.7). Oxygen consumption (VO2) was elevated in hCOX2Tg mice vs. Wt mice during light and dark cycles. As expected, DFU treatment of hCOX2Tg resembled the

Wt situation (Fig. 3A and Supplementary Fig. 6A). Similar results were obtained when

CO2 production was measured and as consequence, EE and RER were also increased in hCOX2Tg mice vs. Wt mice during light and dark periods (Fig. 3B, 3C, 3E and

Supplementary Fig. 6E). However, no differences between genotypes were noted in locomotor activity (Fig. 3D). In agreement with increased cage temperature (Fig. 3F), hepatic COX2derived PGE2 enhanced the expression of thermogenic genes (Ucp1, Page 13 of 47 Diabetes

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Dio2 and Prdm16) in iWAT, suggesting induction of browning. Furthermore,

thermogenicgene expression was also increased in BAT in hCOX2Tg mice (Fig. 4A).

To reinforce these data, we performed a cold acclimation experiment to assess the

differential thermogenic response between Wt and hCOX2Tg mice. As shown in

Figure 4B, coldinduced Ucp1, Dio2 and Prdm16 mRNA levels were significantly

higher in iWAT of hCOX2Tg mice, suggesting a browning process. In BAT, the

differential effects after cold exposure were found in Ucp1 mRNA levels. These data

reinforce the beneficial effects found in energy expenditure in hCOX2Tg mice. Also,

PGE2 was able to induce UCP1 expression in nondifferentiated BAT SVF cells (Fig.

4C).

Hepatic COX2 expression enhances insulin signaling in liver after HFD.

The phosphorylation of Akt and AMPKα were markedly reduced in obese Wt liver as

compared to hCOX2Tg mice (Fig.5A, 5B). These differences in insulin signaling

between Wt and hCOX2Tg mice after HFD could reflect differential expression of

negative modulators of the early steps of insulin signaling. In this regard, PTP1B

protein levels were upregulated in the liver of HFDfed Wt mice compared to similar

genotype fed with RCD, whereas this effect was not observed in hCOX2Tg mice. All

these data suggest that hCOX2Tg mice are protected against insulin resistance under

HFD.

Human and mouse liver cells expressing a COX2Tg are protected against fatty acids

exposure.

To confirm whether liver cells expressing COX2 exhibit a higher insulin sensitivity

and are protected against IR, CHL (human) and NCL (murine) cells with (CHLC,

NCLC) or without (CHLV, NCLV) COX2 expression were treated with 600 µM of

palmitate for 16 h and then stimulated with 10 nM insulin for 15 min. As shown in Diabetes Page 14 of 47

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Supplementary Fig. 7AC, CHLC and NCLC cells per se showed a higher increase in

Akt phosphorylation in response to insulin than CHLV and NCLV cells. Moreover, when cells were treated with palmitate to induce IR, CHLC and NCLC exhibited a higher Akt phosphorylation in response to insulin than CHLV and NCLV reinforcing the results obtained in vivo in hCOX2Tg mice.

DISCUSSION

The present study has demonstrated that constitutive expression of COX2 in hepatocytes protects against steatosis, adiposity, inflammation and hepatic insulin resistance in mice under HFD, implicating the lack of COX2 expression under these circumstances as a novel key player in the development of obesityassociated metabolic dysfunction. Importantly, the leanness of hCOX2Tg mice is associated with increased systemic EE, enhanced thermogenesis and fatty acid oxidation in the liver. Furthermore, pharmacological inhibition of COX2 with DFU reverts to the situation of the Wt mice.

Our previous results (12), by using this transgenic model, demonstrated a protective role of COX2 against liver injury in an experimental model of STZ mediated hyperglycemia. The present study reveals that hCOX2Tg mice exhibit increased insulin sensitivity both in basal conditions and after HFD and are protected against HFDinduced glucose intolerance, steatosis and adiposity. Recent studies suggested that COX2 expression in eWAT protects against obesity by an increase in thermogenic activity (19). Moreover, this effect has been attributed principally to COX

2derived PGE2 (20). In agreement with this, the reduced adiposity found in hCOX2

Tg mice under HFD could be explained as a consequence of elevated EE due to increased substrate consumption. In this way, circulating liverderived PGE2 might be sufficient for the induction of thermogenic genes in adipose tissues, all of these contributing to protection against HFDinduced obesity and improvement of glucose Page 15 of 47 Diabetes

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homeostasis. This was supported by the enhanced thermogenic gene expression found

in iWAT and BAT of hCOX2Tg mice under HFD or cold exposure and by the

induction of UCP1 protein levels in brown preadipocytes stimulated with PGE2.

Elevation of circulating fatty acids and proinflammatory adipokines secreted by

WAT reduces insulin sensitivity in liver and skeletal muscle (21) and adipose tissue

inflammation has been correlated with hepatic steatosis in humans (22). Our results

show that hCOX2Tg mice did not develop the common increase in fat mass and

enhanced adipocyte hypertrophy that is shown in Wt mice under HFD. In agreement

with these data, plasma and hepatic TGs, cholesterol and NEFAs levels were lower in

hCOX2Tg mice and also these mice showed decreased plasma insulin and leptin and

increased adiponectin levels. Moreover, proinflammatory markers were also lower in

hepatic tissue from hCOX2Tg mice vs. with Wt after HFD. Altogether, these

parameters indicate that the beneficial effects on obesity and glucose homeostasis in

hCOX2Tg mice might be due to decreased inflammation.

When Wt and hCOX2Tg mice were fed a HFD we found an important

protection against steatosis and IR vs. Wt mice. Moreover, we found significant

differences in the expression of key enzymes and transcription factors implicated in

lipogenesis and βoxidation, such as Pparg, Scd1, Ppara, Cpt1a and CD36 indicating an

increase in βoxidation in hCOX2Tg mice under HFD. Recent results have implicated

hepatic fatty acid translocase CD36 upregulation with IR and steatosis in nonalcoholic

steatohepatitis and chronic hepatitis C (23). In addition to this, the positive effects of

COX2 on inflammation and IR are evidenced by the prevention of obesity through

increased EE and RER. Diabetes Page 16 of 47

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The PI3K/Akt and AMPK pathways play a central role in integrating diverse survival signals. Moreover, Akt phosphorylation is a critical node of the insulin signaling pathway, being tyrosinephosphorylated IRS1 an upstream mediator of its activation (1). It is known that the Akt pathway is a target of PGs and that Akt phosphorylation is enhanced in the liver of hCOX2Tg mice compared to the Wt, thus reinforcing the survival pathways (12). In Wt liver, HFD impaired insulinstimulated

Akt phosphorylation and this effect was prevented in hCOX2Tg mice. This situation was paralleled in both murine and human derived liver cells overexpressing COX2.

The increase in pAkt/Akt ratio in hCOX2Tg mice under RCD and HFD conditions may be due, besides intracellular signaling, to a direct Akt activation through COX2 dependent PGE2 acting via EP2/EP4 Gβγ dimers as reported by Rizzo et al. (24).

Notably, the effect of PGE2 pretreatment on Akt phosphorylation observed in isolated hepatocytes confirmed that COX2derived prostanoids can be responsible of this effect.

Thus, the improved insulin sensitivity in vivo reflected by hepatic Akt activation found in hCOX2Tg mice might contribute to the amelioration of HFDderived deleterious metabolic effects as exacerbated lipogenesis and hepatic content.

It is known that AMPKα1 protects mice from dietinduced obesity and IR.

AMPKα1 knockdown in mice enhanced adipocyte lipid accumulation, exacerbated the inflammatory response and induced IR (25). We showed that AMPK was phosphorylated in liver cells expressing COX2 under basal conditions and after liver injury (16). Indeed, AMPK phosphorylation and its downstream gene Cpt1a, which regulates a ratecontrolling step of fatty acid oxidation transferring longchain acylCoA into the mitochondria, were increased in hCOX2Tg mice under HFD. Cellular lipid content is determined by the balance between fatty acid oxidation and lipid storage as Page 17 of 47 Diabetes

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TGs. Since the ability of AMPK to induce lipid oxidation is considered an important

feature for insulin sensitization (26), this might also contribute to the beneficial effects

of COX2 expression in hepatocytes on insulin sensitivity.

In addition to the molecular mechanisms mentioned above, differences in insulin

signaling between Wt and hCOX2Tg mice after HFD could reflect differential

expression of negative modulators of the early steps of insulin signaling. Among them,

PTP1B was a likely candidate given its ability to directly dephosphorylate the insulin

receptor and also its expression is induced by inflammatory mediators (27). PTP1BKO

mice are protected against obesityinduced inflammation and peripheral insulin

resistance during aging (28). In agreement with these data, PTP1B protein was up

regulated in the liver of HFDfed Wt mice but not in hCOX2Tg mice, suggesting that

COX2dependent prostaglandins might protect from the elevation of PTP1B during

obesity. In addition, PTP1B modulates leptin sensitivity (29) and decreased leptin levels

are found in hCOX2 Tg mice in both RCD and HFD conditions suggesting a possible

crosstalk between COX2derived signals and leptin.

Henkel et al. reported that PGE2 produced in Kupffer cells interrupt the

intracellular insulin signaling in hepatocytes through serine phosphorylation of IRS1

via EP3receptordependent ERK1/2 activation. Moreover; they demonstrated that in

hepatocytes the EP3 receptor is expressed at higher levels that the other isoforms (30).

However, our results show that the liver of Wt and hCOX2Tg mice expresses

comparable levels of all the EPs receptors (EP1EP4) (not shown) indicating a different

molecular mechanism of action. Our in vivo data in the hCOX2Tg model

characterized by a continuous production of PGE2 in the liver clearly show enhanced

insulinmediated Akt phosphorylation partly due to decreased PTP1B levels under Diabetes Page 18 of 47

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HFD. Therefore, the view emerging from this study is that PGs synthesized by other liver cells (Kupffer or stellate) are not sufficient to cope for the beneficial effects observed when hepatocytes are able to express COX2. In conclusion, the present study demonstrates that expression of COX2 in hepatocytes protects against adiposity, inflammation and hepatic IR in mice under HFD, pointing to COX2 as a potential therapeutic target against obesityassociated metabolic dysfunction. Stable analogues of

PGE2 such as 16,16dmPGE2, resembling COX2 overexpression (31), could be administered to treat steatosis or insulin resistance. In fact, PGE1 and PGE2 and their analogues are used in clinic to improve moderate hypercholesterolaemia among other liver pathologies (32). Furthermore, these results support caution with use of COX inhibitors (COXIBs) in patients with obesity, type 2 diabetes or NAFLD.

ACKNOWLEDGMENTS

The authors have not competing interests

Author contributions

D.E.F., O.M., N.A., A.GR., A. FA., C.C., E.GC., and A.M.V. researched the data.

L.CS. edited the manuscript.

L.B., C.E.C., R.M. and M.C. reviewed the manuscript and contributed to the discussion

A.M.V. and P.MS. wrote and reviewed the manuscript.

The authors thank to Gerardo Pisani (IFISECONICET) Rosario, Argentina, for his technical assistance with the immunohistochemical analysis.

Dr. P. MartínSanz and Dr. A.M. Valverde are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Page 19 of 47 Diabetes

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This work was supported by Financing Program for short stays abroad (CONICET

Argentina) to D.F.; L. CS is supported by a PostDoctoral fellowship from

CONACYT, México; SAF201239732 (MINECO, Spain) and CIBERehd (ISCIII,

Spain) to M.C.; BFU201124760 (MINECO, Spain) to L.B.; S2010/BMD2378

(Comunidad de Madrid, CAM) to L.B. and P.M.S.; RD12/0042/0019 (ISCIII, Spain)

and CIBERehd (ISCIII, Spain) to L.B. and P.M.S; SAF201016037, SAF201343713R

(MINECO, Spain) to P.M.S.; SAF201233283 (MINECO, Spain), S2010/BMD2423

(Comunidad de Madrid, CAM), EFSD and Amylin Paul Langerhans Grant and

CIBERdem (ISCIII, Spain) to A.M.V.

REFERENCES

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inflammation and insulin resistance in skeletal muscle cells. Endocrinology 2010;151:537548 12. Frances DE, Ingaramo PI, Mayoral R, Traves P, Casado M, Valverde AM, Martin Sanz P, Carnovale CE: Cyclooxygenase2 overexpression inhibits liver apoptosis induced by hyperglycemia. Journal of cellular biochemistry 2013; 13. Casado M, Molla B, Roy R, FernandezMartinez A, Cucarella C, Mayoral R, Bosca L, MartinSanz P: Protection against Fasinduced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes. Hepatology 2007;45:631638 14. Llorente Izquierdo C, Mayoral R, Flores JM, GarciaPalencia P, Cucarella C, Bosca L, Casado M, MartinSanz P: Transgenic mice expressing cyclooxygenase2 in hepatocytes reveal a minor contribution of this enzyme to chemical hepatocarcinogenesis. Am J Pathol 2011;178:13611373 15. Trusca VG, Fuior EV, Florea IC, Kardassis D, Simionescu M, Gafencu AV: Macrophagespecific upregulation of apolipoprotein E gene expression by STAT1 is achieved via long range genomic interactions. The Journal of biological chemistry 2011;286:1389113904 16. Mayoral R, Molla B, Flores JM, Bosca L, Casado M, MartinSanz P: Constitutive expression of cyclooxygenase 2 transgene in hepatocytes protects against liver injury. Biochem J 2008;416:337346 17. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, UnalpArida A, Yeh M, McCullough AJ, Sanyal AJ: Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:13131321 18. Hotamisligil GS: Inflammation and metabolic disorders. Nature 2006;444:860867 19. Vegiopoulos A, MullerDecker K, Strzoda D, Schmitt I, Chichelnitskiy E, Ostertag A, Berriel Diaz M, Rozman J, Hrabe de Angelis M, Nusing RM, Meyer CW, Wahli W, Klingenspor M, Herzig S: Cyclooxygenase2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes. Science 2010;328:11581161 20. GarciaAlonso V, LopezVicario C, Titos E, MoranSalvador E, GonzalezPeriz A, Rius B, Parrizas M, Werz O, Arroyo V, Claria J: Coordinate functional regulation between microsomal prostaglandin E synthase1 (mPGES1) and peroxisome proliferatoractivated receptor gamma (PPARgamma) in the conversion of whiteto brown adipocytes. The Journal of biological chemistry 2013;288:2823028242 21. Olefsky JM: Fat talks, liver and muscle listen. Cell 2008;134:914916 22. Cancello R, Tordjman J, Poitou C, Guilhem G, Bouillot JL, Hugol D, Coussieu C, Basdevant A, Bar Hen A, Bedossa P, GuerreMillo M, Clement K: Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity. Diabetes 2006;55:15541561 23. MiquilenaColina ME, LimaCabello E, SanchezCampos S, GarciaMediavilla MV, FernandezBermejo M, LozanoRodriguez T, VargasCastrillon J, Buque X, Ochoa B, Aspichueta P, GonzalezGallego J, GarciaMonzon C: Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in nonalcoholic steatohepatitis and chronic hepatitis C. Gut 2011;60:13941402 24. Rizzo MT: Cyclooxygenase2 in oncogenesis. Clinica chimica acta; international journal of clinical chemistry 2011;412:671687 25. Zhang W, Zhang X, Wang H, Guo X, Li H, Wang Y, Xu X, Tan L, Mashek MT, Zhang C, Chen Y, Mashek DG, Foretz M, Zhu C, Zhou H, Liu X, Viollet B, Wu C, Huo Page 21 of 47 Diabetes

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Y: AMPactivated protein kinase alpha1 protects against dietinduced insulin resistance and obesity. Diabetes 2012;61:31143125 26. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T: Adiponectin stimulates glucose utilization and fattyacid oxidation by activating AMPactivated protein kinase. Nature medicine 2002;8:12881295 27. Zabolotny JM, Kim YB, Welsh LA, Kershaw EE, Neel BG, Kahn BB: Protein tyrosine phosphatase 1B expression is induced by inflammation in vivo. J Biol Chem 2008;283:1423014241 28. GonzalezRodriguez A, MasGutierrez JA, Mirasierra M, FernandezPerez A, Lee YJ, Ko HJ, Kim JK, Romanos E, Carrascosa JM, Ros M, Vallejo M, Rondinone CM, Valverde AM: Essential role of protein tyrosine phosphatase 1B in obesityinduced inflammation and peripheral insulin resistance during aging. Aging Cell 2012;11:284 296 29. Zabolotny JM, BenceHanulec KK, StrickerKrongrad A, Haj F, Wang Y, Minokoshi Y, Kim YB, Elmquist JK, Tartaglia LA, Kahn BB, Neel BG: PTP1B regulates leptin signal transduction in vivo. Developmental cell 2002;2:489495 30. Henkel J, NeuschaferRube F, PatheNeuschaferRube A, Puschel GP: Aggravation by prostaglandin E2 of interleukin6dependent insulin resistance in hepatocytes. Hepatology 2009;50:781790 31. Madsen L, Pedersen LM, Lillefosse HH, Fjaere E, Bronstad I, Hao Q, Petersen RK, Hallenborg P, Ma T, De Matteis R, Araujo P, Mercader J, Bonet ML, Hansen JB, Cannon B, Nedergaard J, Wang J, Cinti S, Voshol P, Doskeland SO, Kristiansen K: UCP1 induction during recruitment of brown adipocytes in white adipose tissue is dependent on cyclooxygenase activity. PloS one 2010;5:e11391 32. Korhonen T, Savolainen MJ, Jaaskelainen T, Kesaniemi YA: Effect of a synthetic prostaglandin E2 analogue, RS86505007, on plasma lipids and lipoproteins in patients with moderate hypercholesterolaemia: efficacy and tolerance of treatment and response in different apolipoprotein polymorphism groups. European journal of clinical pharmacology 1995;48:97102

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Table ΙΙΙ RCD HFD Wt hCOX2Tg Wt hCOX2Tg hCOX2Tg+DFU TG (mg/dl) 23.80 ± 2.70 23.66 ± 4.51 35.78 ± 3.08* 23.28 ± 1.89# 32.59 ± 1.63*† Cholesterol (mg/dl) 101.78 ± 12.98 72.71 ± 10.72 232.17 ± 25.68* 147.78 ± 9.13*# 207.90 ± 15.33*† NEFAs (mEq/l) 18.69 ± 2.21 16.79 ± 2.77 23.44 ± 2.96* 14.17 ± 2.43# 21.69 ± 4.42*† Insulin (ng/ml) 0.94 ± 0.30 0.72 ± 0.17 1.73 ± 0.36* 1.46 ± 0.13* 2.23 ± 0.66* Adiponectin (pg/ml) 7.21 ± 0.72 6.99 ± 1.64 6.08 ± 1.11 8.05 ± 0.35# 4.94 ± 0.61*† Leptin (pg/ml) 7.61 ± 2.07 3.82 ± 0.64* 24.93 ± 1.81* 21.29 ± 1.49*# 24.95 ± 0.89*† Adiponectin/Leptin Ratio 0.95 1.83* 0.24 0.38# 0.20 HOMAIR (A.U.) 19.01 ± 2.89 14.46 ± 0.92# 23.43 ± 0.63 # † PGE2 (pg/ml) 9.43 ± 1.16 14.18 ± 1.10* 8.98 ± 0.35 12.04 ± 0.48* 8.03 ± 0.34

Table I. Biochemical and metabolic characteristics of hCOX2Tg mice after HFD.

Plasma levels of triglycerides, cholesterol, nonesterified free fatty acids (NEFAs) and

insulin, leptin and adiponectin from Wt, hCOX2Tg and hCOX2Tg+DFU mice fed

RCD and after 12 weeks fed HFD. Adiponectin/leptin ratio and HOMAIR. Plasma

PGE2 levels from Wt, hCOX2Tg and hCOX2Tg+DFU mice fed with RCD or HFD

diets. Data are expressed as means ± S.E. for 46 mice of each experimental group.

*p<0.05 vs. WtRCD; #p<0.05 vs. WtHFD, p<0.05 vs. hCOX2TgHFD.

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Table ΙΙΙΙΙΙ

Gene RCD HFD Wt hCOX2Tg Wt hCOX2Tg hCOX2Tg+DFU Tnfa 1 0.65 ± 0.32 2.36 ± 0.79* 1.13 ± 0.38# 1.55 ± 0.66 Il1b 1 1.47 ± 0.30 3.16 ± 1.01* 1.52 ± 0.26# 1.47 ± 0.88 Il6 1 0.44 ± 0.10* 2.16 ± 0.68* 0.63 ± 0.20# 1.67 ± 0.81

Table II. Hepatic mRNA expression of proinflammatory cytokines in hCOX2Tg

mice. Hepatic mRNA levels of proinflammatory cytokines IL6, IL1β and TNFα

measured by real time qPCR normalized to the expression of 36b4 mRNA. Values

represent fold relative to Wt under RCD. Data are expressed as means ± S.E. for 46

mice of each experimental group. *p<0.05 vs. WtRCD; #p<0.05 vs. WtHFD.

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Table ΙΙΙΙΙΙΙΙ

Gene RCD HFD Wt hCOX2Tg Wt hCOX2Tg Cpt1a 1 1.15 ± 0.04 1.70 ± 0.20* 2.87 ± 0.27*# Pgc1a 1 0.68 ± 0.22 1.00 ± 0.13 0.99 ± 0.14 Acox1 1 0.73 ± 0.20 1.92 ± 0.24* 1.18 ± 0.39 Ppara 1 1.61 ± 0.18* 2.54 ± 0.10* 4.41 ± 0.14*# Pparg 1 0.16 ± 0.04* 5.95 ± 0.09* 2.94 ± 0.07*# Acaca 1 0.94 ± 0.18 1.21 ± 0.36 0.89 ± 0.16 Fasn 1 1.59 ± 0.43 1.32 ± 0.72 1.09 ± 0.35 Scd1 1 0.64 ± 0.24 2.25 ± 0.23* 1.46 ± 0.09*# Lipc 1 0.87 ± 0.17 1.05 ± 0.18 1.18 ± 0.16 Lipe 1 0.82 ± 0.08 1.70 ± 0.06* 1.80 ± 0.56 Pnpla2 1 0.97 ± 0.17 1.68 ± 0.56 2.55 ± 0.53 Cd36 1 0.67 ± 0.18 6.30 ± 0.38* 3.65 ± 0.22*# hcox2 1 7365.5 ± 453.8* 1.04 ± 0.43 6749.9 ± 266.2*# mpges1 1 1.58 ± 0.39 0.84 ± 0.35 1.68 ± 0.32*# CyclinD1 1 1.34 ± 0.08* 1.10 ± 0.21 1.51 ± 0.25*# Bcl2 1 1.13 ± 0.11 0.96 ± 0.20 1.57 ± 0.35*#

Table III. Hepatic mRNA expression of genes related to lipogenesis, lipolysis and β

oxidation in hCOX2Tg mice. Hepatic mRNA levels measured by real time qPCR

normalized to the expression of 36b4 mRNA, values represent fold relative to Wt under

RCD. Data are expressed as means ± S.E. for at 46 mice of each experimental group.

*p<0.05 vs. WtRCD; #p<0.05 vs. WtHFD.

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FIGURE LEGENDS

Fig. 1. hCOX2Tg mice are protected from HFDinduced obesity, insulin and

glucose intolerances. Wt and hCOX2Tg mice were fed with HFD ad libitum for 12

weeks. An additional group of hCOX2Tg mice were treated with the COX2 inhibitor

DFU five times in a week during all HFD treatment. (A) Representative photography of

Wt and hCOX2Tg mice. (B) Representative photography of eWAT and liver from Wt

and hCOX2Tg mice (C) Body weight curve of Wt, hCOX2Tg and hCOX2

Tg+DFU mice fed a HFD expressed as % to basal body weight. Data are expressed as

means ± S.E. (n=7 per group). *p<0.05 vs. Wt; #p<0.05 vs. hCOX2Tg. (D) Initial (i)

and final (f) body weight and mean daily food intake. (E) Insulin Tolerance Test (ITT)

after 6 h fasting and (F) Glucose Tolerance Test (GTT) after 16 h fasting of animals

(n=6 per group) after HFD. Graph represents Area Under the Curve (AUC) during GTT.

Data are expressed as means ± S.E. *p<0.05 vs. Wt; #p<0.05 vs. hCOX2Tg.

Fig. 2. COX2 transgenic mice are protected from HFDinduced hepatic steatosis.

(A) Representative images of Hematoxylin & Eosin (H&E)stained liver paraffin

embedded sections from Wt and hCOX2Tg mice under RCD and from Wt, hCOX2

Tg and hCOX2Tg+DFU mice under HFD. (B) Hepatic triglycerides levels in all

experimental groups, measured by enzymatic analysis. Data are expressed as means ±

S.E. (n=7 per group). *p<0.05 vs. WtRCD; #p<0.05 vs. WtHFD. (C) Representative

images of H&Estained sections of paraffinembedded eWAT from Wt, hCOX2Tg

and hCOX2Tg+DFU mice under HFD. (D) Number and adipocyte area expressed in

µm2 of five sections per animal (n=4 per group) (E) Frequency histograms of eWAT

from HFDfed Wt, hCOX2Tg and hCOX2Tg+DFU mice. (F) eWAT+iWAT fat

mass content related to body weight and tibia length. (G) mRNA levels of adipokines

and proinflammatory cytokines measured in eWAT by real time qPCR and normalized Diabetes Page 26 of 47

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to the expression of 36b4 mRNA. Values represent fold relative to Wt under HFD. Data are expressed as means ± S.E. for 46 mice for each experimental group.*p<0.05 vs.

WtHDF; #p<0.05 vs. hCOX2TgHFD.

Fig. 3. Increased energy expenditure in hCOX2Tg mice under HFD. (A) Oxygen consumption (VO2), (B) CO2 release (VCO2), (C) respiratory exchange ratio

(RER=VCO2/VO2), (D) locomotor activity, (E) energy expenditure (EE) and (F) average cage temperature in HFDfed Wt, hCOX2Tg and hCOX2Tg+DFU mice were measured in 12 h light/dark cycles. Data are expressed as means ± S.E. for 4 mice in each experimental group. *p<0.05 vs. WtHDF; #p<0.05 vs. hCOX2TgHFD.

Fig. 4. Enhanced thermogenicrelated genes in hCOX2Tg mice. (A) Ucp1, Dio2 and

Prdm16 mRNA levels were measured in iWAT and BAT by real time qPCR and normalized to the expression of 36b4 mRNA. Values represent fold relative to Wt under

HFD. Data are expressed as means ± S.E. for 6 mice of each experimental group.

*p<0.05 vs. WtHFD. (B). Mice were maintained at 28ºC for one week and after that they were placed at 4ºC for 6 h. Ucp1, Dio2 and Prdm16 mRNA levels were measured in iWAT and BAT by real time qPCR and normalized to the expression of 36b4 mRNA. Results are means ± S.E. for 46 mice of each experimental group. Data are expressed as fold change relative to Wt or hCOX2Tg at thermoneutrality. *p<0.05 vs. cold exposure Wt. (C) BAT precursor SVF cells were stimulated with PGE2 (1 M) or noradrenalin (NA) (1 µM) as a positive control for 24 h and UCP1 expression was analyzed by Western blot. Data are expressed as means ± S.E. for 3 different experiments. *p<0.05 vs. Control without stimulation.

Fig. 5. COX2 expression enhances insulin signaling in liver of mice fed with HFD.

(A) Representative Western blot analysis of Wt and COX2Tg liver under RCD or

HFD; nontreated (Ins) or treated (+Ins) with 0.75 U/kg of insulin (Ins) for 15 min Page 27 of 47 Diabetes

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before euthanasia. (B) Ratios pAkt/Akt and pAMPK/AMPK after densitometric

analysis of Western blot data normalized using βactin as control. Data are expressed as

means ± S.E. for 46 mice of each experimental group. *p<0.05 vs. Wt (Ins); #p<0.05

vs. WtHFD (Ins). Diabetes Page 28 of 47

254x338mm (300 x 300 DPI)

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Supplementary Material

Chemicals.

Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma

Aldrich (St. Louis, MO, USA), Cell Signaling (Boston, MA, USA) and Cayman

Chemical (Ann Arbor, MI, USA). Reagents were from Roche Diagnostics (Mannheim,

Germany) or Sigma Chemical Co. DFU (5,5dimethyl3(3fluorophenyl)4(4

methylsulfonyl) phenyl2(5H)furanone) was from Merck (Rahway, NJ). Human

Regular Insulin (Actrapid) was from Novo Nordisk Pharma S.A (Madrid, Spain).

Reagents for electrophoresis were obtained from BioRad (Hercules, CA, USA). Tissue

culture dishes were from Falcon (Becton Dickinson Labware, Franklin Lakes, NJ,

USA). Tissue culture media were from Gibco (InvitrogenTM, Grand Island, NY, USA).

Cell culture.

The human liver cell line CCL13 (Chang liver, CHL), an immortalized nontumour cell

line derived from normal liver, was purchased from the American Type Culture

Collection (Manassas, VA). Cells were grown on Falcon tissue culture dishes in DMEM

supplemented with 10% fetal bovine serum (FBS) and antibiotics (50 g each of

penicillin, streptomycin and gentamicin per ml) at 37ºC in a humidified air 5% CO2

enriched atmosphere.

Primary culture of adult hepatocytes.

Hepatocytes were isolated from nonfasting male Wt and hCOX2Tg mice by

perfusion with collagenase (1). Cells were plated in 6cm dishes and cultured in

William’s E medium supplemented with 20 ng/ml EGF, 100 U/ml penicillin, 100 µg/ml

streptomycin and 10% FBS for 48 h. Cells were serum starved for 4 h and further

stimulated with 1 or 10 nM insulin for 15 min.

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Brown adipocyte isolation and culture.

Brown fat precursor cells were isolated from the interscapular brown adipose tissue

(BAT) of 20 dayold wildtype mice, as described (2). The process involved successive collagenase digestion, separation of mature adipocytes by flotation and subsequent filtration through 25 m silk filters, obtaining precursor cells by centrifugation. The precursor cells obtained from a pool of 10 mice were cultured in Dulbecco's modified

Eagle's medium (DMEM) supplemented with 3.5 nM insulin, 10 mM Hepes, 50 IU penicillin/ml, 50 g streptomycin/ml, 25 M sodium ascorbate and 10% newborn calf serum (NCS) at 37°C in an atmosphere of 5% CO2 in air with 95% humidity. Once

80% confluence was reached, cells were stimulated with 1 M PGE2, 1 µM noradrenaline (NA) for 24 h and UCP1 expression was analyzed by Western blot.

Characterization of COX-2 vectors. pPyCAGIP was kindly provided by Dr. I. Chambers (University of Edinburgh,

Edinburgh, Scotland). Human COX2 ORF was amplified by PCR from human full length COX2 cDNA cloned into pcDNA1/Amp, a gift from Dr S Prescott (Huntsman

Cancer Institute, Salk lake City, UT, USA). The PCR product was cloned into the XhoI

NotI restriction site of pPyCAGIP vector. The orientation and integrity of the constructs were determined by sequencing.

Generation of stable hCOX2 cells.

Immortalized cell lines were obtained from Wt (NCLV) and hCOX2Tg (NCLC) liver by infection with Large T antigen from SV40 virus. To do this, primary hepatocytes from neonates (35 days old) (NCL cells) were obtained from Wt and hCOX2Tg liver, submitted to collagenase dispersion and cultured as described previously (1). Primary hepatocytes were infected with polybrenesupplemented virus for 48 h and maintained in culture medium for 72 h, before selection with puromycin Page 35 of 47 Diabetes

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for 3 weeks. Then, immortalized cell lines were generated and further cultured for 15

days with argininefree medium supplemented with 10% FBS to avoid growth of non

parenchymal cells. The cells thus generated had hepatocyte phenotype as demonstrated

by the expression of albumin, carbamoyl phosphate synthase and cytokeratine 18 (3).

For plasmid transfections, CHL cells at 70% confluence were exposed for 616 h to

Lipofectamine reagent containing pPyCAGIPhCOX2 or control vector pPyCAGIP. At

the end of this period, the transfection media was replaced with fresh medium

containing 10% FBS, and on the second day after transfection the cells were treated

with medium supplemented with 3 g/ml puromycin. Single colonies of resistant cells

were detectable after 15 days and were selected and subcultured for 30 days.

Subsequent cultures of selected cells were grown in the presence of puromycin. Cells

stably transfected with hCOX2 or empty vectors were termed CHLC and CHLV,

respectively. Different clones were tested with similar COX2 expression.

Palmitate-induced insulin resistance in hepatocytes.

CHL and NCL cells were seeded in a 6well plate (3x105 cells/well) at 70% confluence.

After 24 h, the cells were cultured in 2% FBS and 1% BSA for 4 h, and subsequently

treated with palmitate (600 µM) for 16 h. After 4 h of serumstarving, 1 or 10 nM

insulin was added for 15 min and then cells were collected to analyze insulin signaling.

Fatty acid measurements.

To identify and measure the mobile fatty acid ex vivo, 1H spectra were performed on

Bruker Avance 11.7 Tesla Spectrometer (Bruker BioSpin, Karlsruhe, Germany)

equipped with a 4 mm triple channel 1H/13C/31P HRMAS (High Resolution Magic

Angle Spinning) resonance probe. Samples (15 mg) were introduced into 50 µl zirconia

rotor with 50 µl D2O and spun at 5000 Hz at 4ªC. All spectra were analyzed using the

MestRecnova software (Mestrelab Research S.L.) and the assignments were in Diabetes Page 36 of 47

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agreement with the literature. The profile of saturated fatty acids spectra was analyzed according to the mobility of each chemical group. Six signals resonating at 0.90

[terminal methyls], 1.30 [methylenes] 1.58 [βmethylenes], 2.05 [monoallylic methylenes] and 2.30 [αmethylenes] ppm indicate the presence of saturated fatty acids chains (4). This assay was realized at the Service of Imaging and Spectroscopy by high field magnetic resonance (http://www.siermac.es).

Determination of metabolites, cytokines and hormones.

PGE2 was determined in liver tissue by specific immunoassay (Arbor Assays, Ann

Arbor, MI, USA). Blood glucose levels were measured with an AccuCheck

Glucometer (Roche). Transaminases ALT and AST were assayed spectrophotometrically in plasma with specific kits from BioSystem (BioSystems SA.,

Barcelona, Spain). Triglycerides, cholesterol and nonesterified free fatty acids

(NEFAs) were determined in plasma and liver by enzymatic methods with specific kits from BioSystems and Wako (Wako Chemicals GmbH, Neuss, Germany). Cytokines mRNA levels were measured in liver by qPCR. Plasma insulin, leptin and adiponectin levels were determined by ELISA kits (Millipore, Billerica, MA, USA) following manual instructions. Protein levels were determined with Bradford reagent (BioRad).

RNA isolation.

Total RNA of liver, eWAT, iWAT and BAT was extracted by using TRIzol reagent

(Invitrogen, Carlsbad, CA). Total RNA (1µg) was reverse transcribed using a

Transcriptor First Strand cDNA Synthesis Kit following manufacturer’s indications

(Roche).

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Quantitative real-time PCR analysis.

qPCR was performed with a MyiQ RealTime PCR System (BioRad) sequence

detector using the SsoFast EvaGreen Polymerase method (BioRad) and d(N)6 random

hexamer with the following primers:

Gene Name Sequence Acaca FP: 5' TGGAGCTAAACCAGCACTCCCGA 3' RP: 5' GCTGGATCTCATGTGAAAGGCCAA 3' Acox1 FP: 5' TCGAAGCCAGCGTTACGAG 3' RP: 5' GGTCTGCGATGCCAAATTCC 3' Cd36 FP: 5' AGATGACGTGGCAAAGAACAG 3' RP: 5' CCTTGGCTAGATAACGAACTCTG 3' Cpt1a FP: 5' TCAATCGGACCCTAGACACC 3' RP: 5' CTTTCGACCCGAGAAGACCT 3' Fasn FP: 5' TCGGCGGGTCTATGCCACGA 3' RP: 5' GTCACCCACCTTGGTGCCCG 3' Il1b FP: 5' AGAAGCTGTGGCAGCTACCTG 3' RP: 5' GGAAAAGAAGGTGCTCATGTCC 3' Il6 FP: 5' GAGGATACCACTCCCAACAGACC 3' RP: 5' AAGTGCATCATCGTTGTTCATACA 3' Lipc FP: 5' CCCTGGCATACCAGCACTAC 3' RP: 5' CTCCGAGAAAACTTCGCAGATT 3' Lipe FP: 5' CCAGCCTGAGGGCTTACTG 3' RP: 5' CTCCATTGACTGTGACATCTCG 3' Pnpla2 FP: 5' CAACGCCACTCACATCTACGG 3' RP: 5' GGACACCTCAATAATGTTGGCAC 3' Ppara FP: 5' AGAGCCCCATCTGTCCTCTC 3' RP: 5' ACTGGTAGTCTGCAAAACCAAA 3' Pparg FP: 5' TCGCTGATGCACTGCCTATG 3' RP: 5' GAGAGGTCCACAGAGCTGATT 3 Ppargc1a FP: 5' AAGTGTGGAACTCTCTGGAACTG 3' RP: 5' GGGTTATCTTGGTTGGCTTTATG 3' Scd1 FP: 5' TTCTTGCGATACACTCTGGTGC 3' RP: 5' CGGGATTGAATGTTCTTGTCGT 3' Tnfa FP: 5' CATCTTCTCAAAATTCGAGTGACAA 3' RP: 5' TGGGAGTAGACAAGGTACAACCC 3' Ucp1 FP: 5' TTCAGGGAGAGAAACACCTGC 3' RP: 5' TTCACGACCTCTGTAGGCTG 3' Dio2 FP: 5' GCTTACGGGGTAGCCTTTGA 3' RP: 5' CCAGCCAACTTCGGACTTCT 3' Prdm16 FP: 5' GGCCCAGCACTTGGAATTTG 3' Diabetes Page 38 of 47

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RP: 5' GGCAATGCAGAGTAGAGGCA 3' Adiponectin FP: 5' GTTCCCAATGTACCCATTCGC 3' RP: 5' TGTTGCAGTAGAACTTGCCAG 3' Leptin FP: 5' GAGACCCCTGTGTCGGTTC 3' RP: 5' CTGCGTGTGTGAAATGTCATTG 3' Resistin FP: 5' ACAAGACTTCAACTCCCTGTTTC 3' RP: 5' TTTCTTCACGAATGTCCCACG 3' hcox2 FP: 5' CGCAGTACAGAAAGTATCACAGGC 3' RP: 5' GCGTTTGCGGTACTCATTAAAA 3' mPtges FP: 5' GCCTTTCTGCTCTGCAGCACACT 3' RP: 5' TCGGGGTTGGCAAAAGCCTTC 3' Bcl2 FP: 5' GGAGGCTGGGATGCCTTTGT 3' RP: 5' ACTTGTGGCCCAGGTATGC 3' CyclinD1 FP: 5' GCGTGCAGAAGGAGATTGTGCCA 3' RP: 5' GCACTTCTGCTCCTCACAGACCTCC 3'

Specific primers were purchased from Invitrogen. PCR thermocycling parameters were

95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 10 s. Each sample was run in triplicate and was normalized to 36b4 RNA. The replicates were then averaged, and fold induction was determined in a ∆∆Ct based foldchange calculations.

Histochemistry analysis.

Samples of liver and adipose tissue were fixed in 4% buffered formalin for paraffin preparation. Paraffinembedded liver tissue and eWAT were used for H&E (5).

Western blot analysis.

Extracts from tissue samples (50100mg) or cells (23x106) were obtained as previously described (6). For Western blot analysis, wholecell extracts were boiled for 5 minutes in Laemmli sample buffer, and equal amounts of protein (2030 g) were separated by

1015% SDSpolyacrylamide electrophoresis gel (SDSPAGE). The relative amounts of each protein were determined with the following polyclonal or monoclonal antibodies:

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Antibody Manufacturer Cat No. βactin Santa Cruz Biotechnology A5441 pAkt (Ser473) Cell Signaling Technology 4060 Akt Cell Signaling Technology 9272 pAMPK (Thr 172) Cell Signaling Technology 2535S AMPK Cell Signaling Technology 2532S COX2 Cayman Chemical 160107 pIR (Tyr1158/1162/1163) Merck Millipore 07841 IR Santa Cruz Biotechnology 3020 PTP1B Merck Millipore 07088 UCP1 Abcam ab10983

After incubation with the corresponding antirabbit or antimouse horseradish

peroxidase conjugated secondary antibody, blots were developed by the ECL protocol

(Amersham). Target protein band densities were normalized with βactin. The blots

were revealed, and different exposition times were performed for each blot with a

charged coupling device camera in a luminescent image analyzer (GelDoc, BioRad) to

ensure the linearity of the band intensities. Densitometric analysis was expressed in

arbitrary units.

1 MartinSanz P. Br. J. Pharmacol. 125:1313, 1998

2 Garcia B. Biochim Biophys Acta. 1821:130915, 2012.

3 Valverde AM. Diabetes 52:2239, 2003

4 Righi V. Oncol. Rep. 22:1493, 2009

5 LlorenteIzquierdo C. Am J Pathol 178: 136173, 2011

6 Casado M. Hepatology 45:631, 2007.

Supplementary figure legends

Fig. S1. hCOX-2-Tg mice exhibit an increased insulin sensitivity and glucose

tolerance. Human COX2 protein was only detected in hCOX2Tg mice and, Diabetes Page 40 of 47

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consistently, intrahepatic PGE2 levels were threefold higher compared with Wt mice.

(A) Representative Western blot showing human COX2 expression in liver homogenates from Wt and hCOX2Tg animals using βactin as load control. (B) Intra hepatic PGE2 levels were determined by ELISA in liver homogenates of Wt and hCOX

2Tg mice. Values are the means ± S.E. of four animals per condition. (C) Body weight curve of Wt and hCOX2Tg mice fed a RCD expressed as % to basal weight. Data are expressed as means ± S.E. (n=5 per group). (D) Insulin Tolerance Test (ITT) after 6 h fasting and (E) Glucose Tolerance Test (GTT) after 16 h fasting of RCDfed animals

(n=8 per genotype). Graph represents Area Under the Curve (AUC) during glucose tolerance test. *p<0.05 vs. Wt.

Fig. S2. Effect of COX-2 expression in insulin signaling in hepatocyte primary culture. (A) Representative Western blot analysis (left panel) and densitometric analysis data normalized using βactin as control (right panel) of protein extracts from adult primary hepatocytes from Wt and hCOX2Tg livers treated with 1 or 10 nM insulin

(Ins) for 15 min. Data are expressed as means ± S.E. for 3 different experiments.

*p<0.05 vs. Wt (Ins); #p<0.05 vs. Wt (+Ins). (B) Isolated hepatocytes from Wt and hCOX2Tg livers were incubated for 16 h with 5 µM PGE2 and 5 µM DFU respectively. After starving for 4 h, cells were treated with 1 and 10 nM Ins for 15 min.

Representative Western blot analysis with the indicated antibodies (left panel) and densitometric analysis of the ratios pIR/IR and pAkt/Akt or the protein expression normalized to that of βactin (right panel) are shown. Data are expressed as means ±

S.E. for 3 different experiments. *p<0.05 vs. Wt (PGE2); #p<0.05 vs. hCOX2Tg (

DFU).

Fig. S3. H&E stained sections showing differences in macro- and microvesicular steatosis between Wt and hCOX-2Tg mice. (A) Liver sections stained with H&E from Page 41 of 47 Diabetes

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Wt, hCOX2Tg and hCOX2Tg+DFU mice after 12 weeks fed HFD. The arrows

indicate the occurrence of lipid vacuoles. (B) Steatosis score (A.U.) of experimental

groups under HFD condition. *p<0.05 vs. Wt HFD.

Fig. S4. Plasma levels of transaminases in Wt and hCOX-2Tg mice. ALT and AST

activities were analyzed spectrophotometrically. Data are expressed as means ± S.E. for

4 mice of each experimental group. *p<0.05 vs. Wt RCD; #p<0.05 vs. Wt HFD.

Fig. S5. Analysis of saturated fatty acids in Wt, hCOX-2-Tg and hCOX-2-Tg+DFU

mice. (A) HRMAS MRS were performed to identify and quantify saturated fatty acids

(B) The profile of saturated fatty acids spectra was analyzed. Data are expressed as

means ± S.E. for 46 mice of each experimental group. *p<0.05 vs. Wt HFD, #p<0.05

vs. hCOX2Tg HFD.

Fig. S6. Enhanced metabolism in hCOX-2-Tg mice. Registers of daily oxygen (O2)

consumption (A), carbon dioxide (CO2) elimination (B), respiratory exchange ratio

(RER) (C), activity (D) and energy expenditure (E) of HFDfed Wt, hCOX2Tg and

hCOX2Tg+DFU mice. Data were collected during a 48 h period in a 12/12 lightdark

cycle.

Fig. S7. Human and murine liver cells expressing COX-2 are protected against

palmitate-mediated insulin resistance. Representative Western blot analysis with

indicated antibodies in homogenates from CHLV and CHLC (A) and in NCLV and

NCLC cells (B) incubated with 600 µM palmitate for 16 h followed by the treatment

with vehicle (Ins) or with 10 nM insulin (+Ins) for 15 min. (C) pAkt/Akt ratio after

densitometric analysis of western blot data normalized using βactin as control. Data are

expressed as means ± S.E. for at least 3 independent experiments. *p<0.05 vs. CHLV

or NCLV (Ins); #p<0.05 vs. CHLC or NCLC (Ins). Diabetes Page 42 of 47

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